The present invention relates to a biosensor for facilitating a prompt and highly accurate quantitation of a substrate contained in a sample.
Conventionally, polarimetry, colorimetry, reductometry and other methods using various chromatography have been proposed as a method for quantitative analysis of sugars such as sucrose and glucose. These methods, however, are of poor accuracy, since their specificity toward sugars is not so high. Of these methods, polarimetry, whose operation is rather simple, is greatly influenced by the temperature during the operation, therefore, is not appropriate as a method which enables ordinary people to make an easy quantitation of sugars at home.
Recently, various types of biosensors utilizing specific catalysis of enzymes have been developed.
The following describes quantitation of glucose as an example of quantitating a substrate contained in a sample solution. The method of using glucose oxidase (EC1.1.3.4: hereinafter referred to as xe2x80x9cGODxe2x80x9d) and an oxygen electrode or a hydrogen peroxide electrode is widely known in the art as electrochemical quantitation of glucose (for example, xe2x80x9cBIOSENSORxe2x80x9d edited by Shuichi Suzuki, Kodan-sha).
GOD selectively oxidizes substrate xcex2-D-glucose to D-glucono-xcex4-lactone, using oxygen as an electron mediator. In the presence of oxygen, oxygen is reduced into hydrogen peroxide during this oxidation by GOD. The decreased amount of oxygen is measured by the oxygen electrode, or otherwise, the increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. Since both the decreased amount of oxygen and the increased amount of hydrogen peroxide are in proportion to the amount of glucose contained in a sample solution, the quantitation of glucose can be made from either of these amounts.
As inferred from the reacting process, this method has a defect that the results of measurement are largely affected by the concentration of oxygen contained in the sample solution. Moreover, the measurement becomes impossible in case of the absence of oxygen in the sample solution.
Therefore, a new type of glucose sensor substituting an organic compound such as potassium ferricyanide, a ferrocene derivative or a quinone derivative or a metal complex for oxygen as an electron mediator has been developed. With this type of sensor, by oxidizing a reductant of the electron mediator produced as the result of the enzyme reaction on the electrode, the concentration of glucose contained in the sample solution can be obtained based on the oxidation current. The substitution of such an organic compound or a metal complex for oxygen as an electron mediator makes it possible to form a reaction layer wherein known amounts of GOD and the electron mediator therefore are stably and accurately carried on the electrode. In this case, since the reaction layer, with its condition almost dry, can also be integrated with the electrode system, a disposable glucose sensor based on this art has recently drawn a lot of attention. The typical example of this sensor is the biosensor disclosed in Japanese patent publication No. 2517153. The disposable glucose sensor facilitates easy measurement of glucose concentration by simply introducing a sample solution into the sensor which is detachably connected to a measuring device. This method is applicable to not only quantitation of glucose but quantitation of any other substrate contained in the sample solution.
In the measurement using above-mentioned glucose sensor, the concentration of substrate contained in the sample solution can easily be detected with a small amount of sample solution (several xcexcl). However, a high-performance, easy-to-handle biosensor which would enable measurement with a much smaller amount, in particular 1 xcexcl or less of sample solution is anxiously expected to be developed in various fields in recent years.
Also, a conventional electrochemical glucose sensor comprises an electrode system disposed on single plane in most of the cases. If the electrode system is on single plane and an extremely small amount of sample solution is used, the resistance to charge-transfer between electrodes, mainly ion transfer, is increased so that a variance in the results of the measurement may be caused.
In order to solve above problems, the biosensor in accordance with the present invention comprises a working electrode base plate, a counter electrode base plate and a reagent layer containing at least an enzyme and an electron mediator, wherein a working electrode disposed on said working electrode base plate and a counter electrode disposed on said counter electrode base plate are positioned so as to mutually face having a space therebetween.
In other words, the working electrode and the counter electrode are opposed to each other via an opening space. To form such an opening space, at least one of these base plates may have a curved portion, a concave portion or the like.
The present invention provides a biosensor comprising a working electrode base plate, a counter electrode base plate, a spacer member placed between said both base plates and a reagent layer containing at least an enzyme and an electron mediator, wherein a working electrode disposed on said working electrode base plate and a counter electrode disposed on said counter electrode base plate are positioned so as to mutually face with a spacer member placed therebetween.
In this case, it is preferable that at least one of said working electrode base plate and said counter electrode base plate has a through-hole which exposes an electrode terminal of the other plate to outside.
Namely, when the working electrode base plate has the through-hole, an electrode terminal of the counter electrode is exposed to outside. And, when the counter electrode base plate has the through-hole, an electrode terminal of the working electrode is exposed to outside. Of course, both base plates may have the through-holes.
It is preferable that one of said working electrode base plate and said counter electrode base plate has a cut-away portion which exposes an electrode terminal of the base other plate to outside and that a lead connected to the electrode on a surface of the base plate having a cut-away portion extends, via a side surface of the base plate having the cut-away portion, to the back of the surface where the lead is connected.
Also, it is preferable that one of said working electrode base plate and said counter electrode base plate has a through-hole filled with a conductive material and a cut-away portion which exposes an electrode terminal of the other base plate to outside and that a lead connected to the electrode on a surface of the base plate having the cut-away portion extends, via the conductive material, to the back of the surface where the lead is connected.
The present invention provides a biosensor comprising an insulating base plate provided with a groove on its surface, a cover member jointed to said insulating base plate to form a space for accommodating a sample in said groove, a working electrode and a counter electrode disposed so as to mutually face in said groove and a reagent layer containing at least an enzyme and an electron mediator disposed in said groove.